Many amino acids occur in nature and are characterized by having at least one carboxyl group and one amino group separated by one or more carbons. Peptides are condensation products resulting from elimination of water between the carboxyl group of one amino acid and the amino group of another. In addition, other reactive groups may be present in the individual amino acids.
Because of the extraordinary high unit value of some peptides, such as peptide hormones and peptide vaccines, this area of chemistry has received a great deal of attention. Some synthetic peptides, such as reaction catalysts and food additives like the artificial sweetener, Aspartame, have also become important industrial specialty chemicals.
The chemical preparation of a peptide is accomplished either by solution chemistries or by solid phase chemistries. The chemistries of the two methods are quite similar. Both require a sequence of reactions involving the coupling of a protected amino acid with a peptide chain, deprotecting the newly-coupled amino acid, and with some chemistries, neutralization. Solution chemistry differs from solid phase chemistry primarily in the methods use for isolation and purification of the chemical intermediates. In a solution synthesis, the intermediates are usually isolated by precipitation or evaporation of the solvent. The purifications are done by such techniques as recrystallization, precipitation, and chromatography. On the other hand, in a solid phase synthesis, in which the growing peptide chain is attached to the solid substrate, the isolation is done by filtration, and the purifications are done by washing with solvent and filtration. Because these steps are repetitive, the solid phase method can be automated.
A typical solid phase peptide synthesis is often referred to as the Merrifield Method after its inventor Dr. Bruce Merrifield of Rockefeller University. The Merrifield process starts with an amino acid coupled via the alphacarboxyl group to a suitable resin. The alpha-amino group of the carboxyl terminal amino acid of the peptide being protected by a t-butyloxycarbonyl (BOC) group which reduces the nucleophilicity of the nitrogen. The coupling of each subsequent amino acid incorporates the following reaction scheme:
1. Removal of the BOC group with 50 percent trifluoroacetic acid (TFA) in dichloromethane. This step is often performed in two stages: a short (5 minute) deprotection to condition the resin, and a long (20-30) minute treatment to insure complete removal or deprotection. ##STR1##
2. Washing the resin with dichloromethane to remove all traces of TFA. This washing step is usually repeated 3 or 4 times.
3. Neutralizing with 5 percent triethylamine (TEA) in dichloromethane in order to deprotonate the alpha-amino terminus resulting from the deprotection step so that the subsequent nucleophilic displacement coupling reaction will proceed. This step is usually performed twice to insure complete neutralization. ##STR2##
4. Washing the resin with dichloromethane 3 or 4 times to remove excess TEA and TEA salts.
5. a) Adding 3-fold molar excess of a BOC-protected amino acid dissolved in dichloromethane to the reaction mixture followed by an equivalent amount of a carboxyl group activating reagent such as dicyclohexylcarbodiimide (DCC). The coupling reaction is nearly complete in 15 minutes or less but peptide chemists often let the reaction run for an hour or more to insure complete reaction. ##STR3##
6. Washing the resin ester with dichloromethane 3 or 4 times to remove excess reagents and by-products from the chemical reaction. One by-product, dicyclohexylurea (DCU), is insoluble in dichloromethane and is often removed by washing the system with 3 or 4 volumes of methanol. The methanol washes are followed by 3 or 4 dichloromethane washes to prepare the system for the next cycle.
Even though this coupling method is very effective, it is costly because one equivalent of reagent is wasted for each equivalent that couples. To reduce this waste, some researchers add an alcohol, such as N-hydroxyl-benzotriazole, that forms an active ester of the amino acid.
Because this intermediate is unstable and rearranges to a very stable acylurea which consumes active reagents, many scientists modify this reaction scheme. ##STR4## If only one-half equivalent of protected amino acid is used, an active symmetric anhydride is formed. ##STR5## Most automated peptide synthesizers also perform the symmetric anhydride and active ester chemistries automatically. One commercial machine, sold by Applied Biosystems, Inc., does these chemistries in a manner described in U.S. Pat. No. 4,668,476. This on-machine activation is a major portion of the cost of the construction of such machines.
The Merrifield BOC solid phase method depends upon having a C-terminal amino acid resin linkage that is stable to mild acid such as trifluoroacetic acid and unstable to (cleaved by) strong acids such as anhydrous hydrofluoric acid.
An alternative procedure for solid phase synthesis relies on a base-labile protecting group on the alpha-amino group, such as flourenylmethoxycarbonyl (FMOC), instead of the acid-labile BOC group. By using this type of protection, the linkage between the C-terminal amino acid and the resin support can be labile to mild acids like trifluoroacetic acid. This then eliminates the need for using anhydrous hydrofluoric acid at the end of the synthesis.
The general protocol for the addition of one amino acid to the peptide chain using the base labile deprotection scheme is as follows:
1. Removal of the FMOC protecting group with 20 percent piperidine in dimethylformamide (DMF). ##STR6##
2. Washing the resin with pure DMF 3 or 4 times to remove all traces of reagents, side products and piperidine.
3 Adding an activated FMOC amino acid. ##STR7##
4. Washing the resin 3 or 4 times with pure DMF to remove any excess FMOC amino acid ester and reaction by-products.
5. Repeating this cycle until the desired peptide chain has been constructed.
Even though this procedure is simplified, an activated amino acid is still required as the BOC procedure described above and the same cost factors apply. Pre-formed activated amino acids (e.g. pentafluorophenyl esters) are stable in dry form but are not as reactive as the symmetric anhydrides described above, thus product yield is lower and reaction times are longer.
An alternative activation method that has many advantages over the DCC methods described above has been used for the base labile FMOC approach by the inventors and others (Fournier, et al.; 1987 Peptide Symposium Proceedings), which utilizes benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP or Castro's reagent), as the activating agent instead of DCC: ##STR8## In this method, there are no insoluble by-products formed. The chemical intermediate is more reactive and better coupling yields have been reported. However, because the reagent is not very stable in solution, new reagent must be prepared for the machine each day or two. This prevents long term unattended automated synthesis which reduces the main advantages of an automated peptide synthesizer.